In this project, we constructed three active site interface PR mutants (PRT26A, PRD29N, and PRR87K) and two termini interface PR mutants (PR1-C95A and PR97/99) and determined their ESI-MS profiles. In the ESI-MS spectra, the peaks of PRD29N monomers ([PRD29N]6+ and [PRD29N]7+) were greater than those of PRWT ([PRWT]6+ and [PRWT]7+). The peaks of PRD29N monomers ([PRD29N]6+ and [PRD29N]7+) were also greater than those of PRD29N dimers ([PRD29N]9+ and [PRD29N]11+). The same was true for the cases of PR1-C95A and PR97/99. These finding suggest that the dimers represent small components in the three mutated PR species. However, as assessed using the FRET-based HIV-1 expression assay, PRD29N dimerization was apparently completely disrupted. Of note, the FRET-based system determines the FRET signal (CFP fluorescence after photobleaching/CFP fluorescence before photobleaching: CFPA/B ratio) in one cell at one time, accumulates approximately 20 to 30 cells' data, and obtains the average of the CFPA/B ratios. Then, if the average value is greater than 1.0, it is judged that FRET occurred, indicating that PR dimerization took place within the cell. However, there is variability in the CFPA/B ratios within the assay data, probably due to, but not limited to (i) unequal expression of PR proteins; (ii) uneven occurrence of protein-protein interactions (i.e., dimerization); and (iii) differing compartmentalization of the expressed PR species within the transfected cell population. Nevertheless, the FRET-based HIV-1 expression assay system only calls whether FRET occurred or did not occur. Thus, the FRET-based HIV-1 expression assay system inherently fails to identify the presence of a small amount of dimers or monomers. Therefore, in the case of PRD29N, the FRET signal was determined as not detected. However, the ESI-MS system we used in the present study directly and more quantitatively identifies PR monomers and dimers. Thus, the ESI-MS system correctly recognized both a major fraction of PRD29N monomers as well as a minor fraction of PRD29N dimers. Accordingly, increasing the expression of PRD29N by increasing the amount of plasmid for transfection does not increase the specific FRET-based signal, which we have confirmed in our initial conditioning phase of the construction of the system. Both PRT26A and PRR87K failed to dimerize as examined with the FRET-based HIV-1 expression assay and no dimerized PR species were seen in the ESI-MS spectra. When we examined the ESI-MS spectra of PR1-C95A and PR97/99, the peaks of +6 charged monomer ions were much greater than in the PRWT spectrum; however, PR dimer species were also present. Moreover, DSF analysis showed that PR1-C95A and PR97/99 dimers were unstable. Taken these data together, it is strongly suggested that the protease dimerization process undergoes two steps: (i) initial albeit weak intermolecular interactions occurring in the active site interface and (ii) subsequent interactions occurring in the termini interface, resulting in the complete and tight protease dimerization. In the present study, we also demonstrated that DRV binds to PRWT monomers as well as dimers, while other conventional PIs including SQV and NFV bind only to dimers, confirming that DRV uniquely has dual activity against PRWT: inhibition of PRWT dimerization and proteolytic activity as previously described. It is also noteworthy that the present data clearly showed that DRV binds to PRWT monomer subunit in a one-to-one molar ratio. We have previously selected highly DRV-resistant HIV-1 variants (HIVDRVR) and identified that HIVDRVR had acquired a unique combination of four amino acid substitutions (V32I/L33F/I54M/I84V) in the proximity of the active site interface of its PR. In the present data, DRV virtually completely failed to bind to PR32/33/54/84 monomers and only a small amount of DRV-bound PR32/33/54/84 dimers was identified. However, L97A and F99A substitutions did not affect DRV's monomer and dimer binding. These results indicate that the binding domain in PR monomers for DRV is located distantly from the termini interface and is close to the active site interface, in line with the results of computational results reported by Huang et al. Thus, the present ESI-MS analysis results strongly suggest that DRV blocks the first step of PR dimerization process involving the active site interface, by binding to PR monomers in a one-to-one molar ratio. We have shown that once stable PRWT dimers are formed, DRV no longer disrupts the dimers as examined using the FRET-based assay. To ask whether DRV's binding to PRWT monomers yields sufficient force to block PRWT dimerization, the determination of the binding affinity of DRV for the folded monomers seems to be technically highly challenging. However, the present ESI-MS data showed that the amount of DRV-bound PRWT monomers appeared to be greater than that of DRV-bound PRWT dimers. Moreover, we have determined the thermal stability of DRV-bound PRWT using DRV-bound PRWT, which appears to contain more DRV-bound PRWT monomers than DRV-bound PRWT dimers, turned out to be highly stable. The DSF data also suggested that DRV's binding to PRWT monomers should have sufficient force to block PRWT dimerization, inhibiting the formation of transient dimers. It is of note that if the formation of transient dimers (1st step of the dimerization process) is blocked by DRV, the formation of stable dimers through the termini interface interactions (2nd step of the dimerization step) no longer occurs. The determination of the exact binding site of PR monomer for DRV awaits further investigation such as crystallographic analysis of PR monomer complexed with DRV and other dimerization inhibitors. A few groups have reported protease dimerization inhibitors targeting the terminal interface of PR. However, none of such inhibitors have been of clinical utility probably since PR dimers, once formed, are highly stable to de-dimerize with the potent dimerization forces in the termini interface. On the other hand, the active site interface interactions play a critical role for PR dimerization, but the dimers formed are thought to be relatively unstable. Thus, the development of new dimerization inhibitors targeting the active site interface would be highly suitable. It is also noteworthy that the ESI-MS approach is more quantitative than the FRET-based HIV-1 expression system, and we demonstrated two features: (i) DRV binds to PRWT monomers and dimers, while (ii) DRV binds only to TFR-PRD25N monomers. Thus, ESI-MS analysis is useful in analyzing how PR monomers and dimers act in the presence or absence of dimerization-targeting drugs. The new findings demonstrated in the present study should help understand the mechanism of HIV-1 protease inhibition and should also help develop novel and more potent protease inhibitors.